CN102558362A - Fusion protein for treating diabetes and preparation method for fusion protein - Google Patents
Fusion protein for treating diabetes and preparation method for fusion protein Download PDFInfo
- Publication number
- CN102558362A CN102558362A CN2012100378514A CN201210037851A CN102558362A CN 102558362 A CN102558362 A CN 102558362A CN 2012100378514 A CN2012100378514 A CN 2012100378514A CN 201210037851 A CN201210037851 A CN 201210037851A CN 102558362 A CN102558362 A CN 102558362A
- Authority
- CN
- China
- Prior art keywords
- fusion rotein
- mutants
- exendin
- linker
- igg1
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to the technical field of medicines for treating diabetes, in particular to a fusion protein for treating diabetes and a preparation method for the fusion protein. The fusion protein is recombined by 2 to 8 Exendin-4-Linkers and a human IgGFc mutant; Linker is a flexible peptide fragment; and the human IgGFc mutant is an IgG1Fc, IgG2Fc or IgG4Fc mutant. The invention also discloses the preparation method for the fusion protein. The recombined fusion protein obtained by the steps of performing high-level expression in Chinese hamster ovary (CHO) cells, affiliating, and performing ion exchange and molecular sieve chromatography has the bioactivities of stimulating the secretion of insulin and inhibiting glucagon generated after dinner from being released which are possessed by Exendin-4, also has the characteristics of prolonging the half-life period of the Exendin-4 in serum, is used for treating type I and type II diabetes, can effectively reduce the psychological and physiological burden of patients, is safe in use and high in practicability, and can be massively produced and sold.
Description
Technical field
The present invention relates to the Remedies for diabetes technical field, particularly a kind of fusion rotein that is used to treat mellitus and preparation method thereof.
Background technology
Mellitus are a kind of serious self metabolism disorder property diseases, can cause that serious cardiovascular function is disorderly, have characteristics such as high incidence and high mortality, and serious harm human health and life quality have caused heavy economical load with social for patient.According to the epidemiology survey statistics, present global diabetic subject's sum has exceeded 1.2 hundred million, and wherein about 90% is type II diabetes, and this numeral also will rise steadily, and estimate 2025, and world wide will have 300,000,000 people to suffer from mellitus.Therefore, the exploitation of special efficacy diabetes medicament has very high social benefit and market outlook.
IDDM is mainly treated by insulin injection at present; The treatment of type II diabetes is main with OHA then, comprises the plain medicine of sulphur urea, biguanides, alpha-glucosidase inhibitor, thiazolidine diketone derivative class and insuline pro injection etc., and the main drawback of these medicines is that untoward reaction is bigger; Life-time service can produce drug resistance; Can cause hypoglycemic danger like Regular Insulin, other oral pharmaceutical can cause main organs damages such as liver, kidney, and final patient can cause the treatment failure to medicine generation tolerance; And now mostly be two or three medication combined medication, increased patient's body and economical load.Therefore, utilizing novel targets and new treatment to develop more safe and effective diabetes medicament, as not causing hypoglycemia and can improving patient β cell function etc., is the focus of diabetes medicament research.
At present, as a kind of new mechanism and target spot, be that basic novel diabetes medicament is paid close attention to by increasing biological study mechanism and drugmaker with the incretin.(incretin-Glucagon-like-peptide-1 is to stablize one of crucial regulatory factor of insulin level and hyperglycemic-glycogenolytic factor level in the body GLP-1) to glucagon-like peptide.Exendin-4 is isolated a kind of 39 amino acid whose polypeptide that contain from South America Monster (Gila monster Heoderma suspectrum) oral secretion, has 53% homology with endogenous GLP-1, and biological activity is similar with endogenous GLP-1.And; Because the 2nd of Exendin-4 is glycocoll, rather than the L-Ala of GLP-1, Exendin-4 is not decomposed by dipeptidyl peptidase (DPP-IV) after getting in the body influence; GLP-1 is only 1～2 minute transformation period in vivo; Promptly decomposed by DPP-IV, than GLP-1, the EDD of Exendin-4 is longer.On diabetes animal model, it is all effective that no matter oral, sublingual administration, lung, air flue still are that nasal cavity gives Exendin-4.
GLP-1 receptor stimulant Exendin-4 has unique mechanism of action aspect the mellitus in treatment: it relies on mode regulating and controlling blood sugar level with sugar, promptly stimulates secretion of insulin during hyperglycemia, and does not stimulate secretion of insulin during hypoglycemia.Therefore, compare with other mellitus class medicines and have a lot of advantages: 1. can significantly reduce glycolated hemoglobin level and glucose level control; 2. because its adjusting function relies on glucose level, therefore can not cause hypoglycemic generation; 3. strengthen satietion, reduce excessive absorption of food and fat-reducing; 4. improve the β cell function; 5. control the generation of postprandial hyperglycemia.Feed back from present clinical data, untoward reaction is slight, has improved drug safety greatly.The comparatively outstanding Byetta (synthetic Exendin-4) that comes company like: gift of this target spot research in 2005 through FDA approval listing, the Li Lalu peptide of Novo Nordisk Co.,Ltd (Liraglutide, it be the GLP-1 simulating peptide) went on the market through the FDA approval in January, 2010.A large amount of experimental datas and clinical data are verified, and GLP-1 and analogue Exendin-4 thereof are safe and effective as novel Remedies for diabetes.
Though Exendin-4 has BA identical with GLP-1 and advantage (as previously mentioned), and has the transformation period of being longer than GLP-1, its transformation period is still shorter, and patient needs to inject twice every day, makes troubles to patient in actual use.Therefore, develop the focus that more long lasting diabetes medicament is present this area research based on GLP-1 and GLP-1 receptor stimulant Exendin-4.
Prolonged drug research is in recent years by biological technical field scientist extensive concern.People prolong the transformation period in the body of polypeptide drugs through variety of way, domestic method as: Biodegradable material sustained release preparation, amino acid mutation/modification protease inhibitor two mutants, the modification of polypeptide PEGization, recombination fusion protein as merge with human serum albumin, with the fusion of immunoglobulin Fc segment etc.
(immunoglobulin G is one of albumen the abundantest in the blood of human body IgG) to the IgG immunoglobulin like protein, and its transformation period can reach 21 days.Confirm that at present IgG molecule long half time in vivo combines back recycling relevant with the mode that its Fc section and newborn Fc acceptor (FcRn) rely on pH.At present, prolong the pharmaceutical grade protein transformation period many successful examples listings have been arranged to merge with IgG molecule Fc section, like Nplate, Orencia, Enbrel, Arcalyst, Amevive etc.The quasi-drugs (the benefit match is general) that China SFDA has also ratified a kind of Enbrel is used for treating rheumatoid arthritis and relative disease thereof.These have confirmed further that all protein and IgG molecule Fc section amalgamation and expression are to improve the drug half-life efficient ways.
The IgG immunoglobulin like protein mainly is divided into four hypotypes, is respectively IgG1, IgG2, IgG3 and IgG4, and wherein the highest at people's in-vivo content with the IgG1 type especially, its molecular structure such as Figure of description are shown in Figure 1.
Also have in the prior art and adopt hyperglycemic-glycogenolytic factor similar peptide GLP-1-1, GLP-1 two mutants or be used for prevention and treatment I type and type II diabetes with the fusion rotein of the same merit factor (Exendin-4) of its at least 50% homology and IgG Fc.Yet; The Fc zone of human normal immunoglobulin has important BA in vivo; Combine with the Fc acceptor (Fc γ Rs) of cell surface like (1), cause coming effectively to kill and wound pathogenic agent or malignant cell through ADCC effect (ADCC effect); (2) partly combine with the first complement component C1 q, the cytotoxic effect (CDC effect) that causes the complement dependence effectively kills and wounds pathogenic agent; (3) long half-lift of the acquisition through recycling after combining with newborn Fc acceptor (FcRn) thereby with the mode that pH relies on.When improving the transformation period as the medicine fusion mode, ADCC effect and CDC effect are useless in the segmental biological effect of IgG Fc, or even deleterious, tend to cause the generation of untoward reaction.In addition; What the fusion of GLP-1 receptor stimulant GLP-1 or Exendin-4 and IgG was adopted in the prior art is that a purpose peptide section and IgG merge, and its specific activity is relatively low, and effective dose is bigger than normal; Be prone to cause the untoward reaction risk to improve, production cost is also higher simultaneously.
Summary of the invention
The objective of the invention is to the deficiency of prior art and a kind of have 2-8 Exendin-4-Linker flexibility peptide section of long-actingization characteristic and the fusion rotein that is used to treat mellitus that human IgG Fc two mutants reassembles into are provided; Its transformation period obviously prolongs; Compliance is good; Effectively reduce patient's physiology and psychological burden, safe in utilization and practical.
Another object of the present invention is to treat being used to of providing to the deficiency of prior art that a kind of flexible peptide section of 2 Exendin-4-Linker with long-actingization characteristic and human IgG Fc two mutants reassemble into the preparation method of the fusion rotein of mellitus; Its technical maturity; Help realizing the industrialization production and selling, the fusion rotein transformation period that makes obviously prolongs, and compliance is good; Effectively reduce patient's physiology and psychological burden, safe in utilization and practical.
For realizing above-mentioned purpose, the present invention adopts following technical scheme.
A kind of fusion rotein that is used to treat mellitus is provided, and said fusion rotein is fusion rotein N (Exendin-4-Linker)-human IgG Fc two mutants that N Exendin-4-Linker and human IgG Fc two mutants reassemble into;
Wherein, Linker is one group of flexible peptide section of being made up of hydrophobic amino acid, and its amino acid whose number is less than 25;
Wherein, human IgG Fc two mutants is IgG1 Fc, IgG2 Fc or IgG4 Fc two mutants;
Wherein, N is 2-8.
Exendin-4, it is for carrying out the main pharmacodynamics target spot of treatment mellitus, and its biological activity is more stable, is difficult for decomposing, and through promoting the release of Regular Insulin with intravital GLP-1 receptors bind, thereby reaches the purpose of treatment mellitus.
The nucleotide sequence of Exendin-4 is shown in SEQ ID NO:1.The aminoacid sequence of Exendin-4 is shown in SEQ ID NO:2.
Linker (connection peptides) is one group of flexible peptide section of being made up of hydrophobic amino acid; It mainly acts on is that Exendin-4 molecule and IgG Fc two mutants are connected and reduce sterically hindered between Exendin-4 functional zone and the IgG Fc two mutants functional zone better; Avoid the activity of Exendin-4 being descended, thereby farthest guarantee Exendin-4 and receptors bind and bring into play its BA owing to directly connecting the space steric effect that causes.
Linker; Generally be 5-15 the amino acid flexible peptide section of (perhaps being less than 25 amino acid); The existence of Linker is for after preventing that each active region from directly connecting; Owing to space steric effect makes each active zone can not be well and corresponding receptors bind, and the activity that causes descends, so the existence of Linker is in theory than there not being Linker more can keep the active function of each active zone well; The length of Linker also has certain limitation so, and the document that has delivered in this field supports that generally amino acid length is less than in 25 amino acid scopes.
IgG Fc two mutants is the major function district that the present invention reaches long-acting purpose, its mainly act on be through Exendin-4 with after it is connected, increase the molecular weight of polypeptide, thereby reduce the filtration of renal glomerulus; Simultaneously because the Fc section can prolong the transformation period of Exendin-4 effectively with FcRn receptors bind in the human body; In order to reduce unnecessary ADCC effect and the CDC effect that natural IgG Fc section is brought, the present invention has carried out corresponding sudden change according to the aminoacid sequence of minimum (being that ADCC effect and CDC effect are minimum) the IgG2 type Fc section of lytic activity in the IgG hypotype simultaneously.
The mechanism of action of the present invention is: Exendin-4 and IgG Fc two mutants merge the molecular weight that can effectively improve polypeptide; Avoided unnecessary ADCC effect and CDC effect, IgG Fc section can be through prolonging its transformation period in vivo with intravital FcRn receptors bind simultaneously.
Further, said fusion rotein is the fusion rotein that 2 Exendin-4-Linker and human IgG1 Fc two mutants reassemble into.Because 2 Exendin-4-Linker molecules are arranged, under equal volumetric molar concentration, this fusion rotein shows the BA higher than single Exendin-4-Linker.
Wherein, because IgG1 is that content is maximum in the human body, consider that from the angle of safety said human IgG Fc two mutants is preferably IgG1 Fc two mutants; The amino acid whose sudden change position of IgG1 Fc two mutants is the combinatorial mutagenesis position of among the Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236/Ala327Gly/Ala330Ser/Pro331Ser one sudden change position or at least two positions; (the amino-acid residue numbering is by the described EU number system of people such as Kabat " SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST " the 5th edition to the Position Number that wherein suddenlys change according to the EU numbering system; United States Department of Health and Human Services, 1991).
Particularly, the sudden change position of human IgG1 Fc two mutants can be the combinatorial mutagenesis position of four positions of Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236, the unnecessary ADCC effect that it can effectively avoid natural IgG Fc section to bring.The sudden change position of human IgG1 Fc two mutants also can be the combinatorial mutagenesis position of three positions of Ala327Gly/Ala330Ser/Pro331Ser, the unnecessary CDC effect that it effectively avoids natural IgG Fc section to bring.
Further preferably, the sudden change position of human IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of seven positions of Ala327Gly/Ala330Ser/Pro331Ser, unnecessary ADCC effect and CDC effect that it can effectively avoid natural IgG Fc section to bring.The nucleotide sequence of its IgG1 Fc two mutants is shown in SEQ ID NO:3.
The codon position of amino acid mutation position be according to the nucleotide sequence contrast of natural IgG1 Fc; But; Because this sequence is the sequence after the codon optimization; Though the amino acid that each codon its appropriate translation is come out also with natural identical (except the sudden change position), whole Nucleotide has been optimized for and has been most appropriate in mammal cell CHO, express.The technology of knowing according to the industry so; Even the natural acid sequence of above-mentioned each functional zone is perhaps through the nucleotide sequence after the different modes appropriateness optimizing codon; That perhaps carries out to different expressive hosts such as intestinal bacteria, yeast, insect and other mammalian cell is codon optimized; As long as its corresponding amino acid do not change, all should be regarded as identically with the present invention, all belong to protection scope of the present invention.
The aminoacid sequence of its IgG1 Fc two mutants is shown in SEQ ID NO:4.
Amino acid after the sudden change and position thereof are Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to the EU numbering system.
Certainly, the sudden change position of human IgG Fc two mutants can be any position or any two positions or the above bonded combinatorial mutagenesis position, any two positions in above-mentioned seven positions.
When the aminoacid sequence of fusion rotein of the present invention was represented with 4 Exendin-4-Linker molecules, this fusion rotein was 4 (Exendin-4-Linker)-human IgG Fc two mutants, and Linker is respectively (Gly when connection peptides
4Ser) 3 with (Gly
4Ser) 1 o'clock, and human IgG Fc two mutants is IgG1 Fc two mutants, its aminoacid sequence is shown in SEQ ID NO:5.
When the aminoacid sequence of fusion rotein of the present invention was represented with 6 Exendin-4-Linker molecules, this fusion rotein was 6 (Exendin-4-Linker)-human IgG Fc two mutants, and Linker is respectively (Gly when connection peptides
4Ser) 3 with (Gly
4Ser) 1 o'clock, and human IgG Fc two mutants is IgG1 Fc two mutants, its aminoacid sequence is shown in SEQ ID NO:6.
Therefore, can the rest may be inferred goes out the structural formula of fusion rotein N (Exendin-4-Linker)-human IgG Fc two mutants that the present invention protects, N=2-8 according to top example.
Wherein, said Linker is flexible peptide section (Gly
4Ser) n, n=1-5 or flexible peptide section (Ala
3Ser
2) n, n=1-5.Certainly, the Linker that other that the flexible peptide section Linker that adopts among the present invention can also be this area are commonly used.
Linker is flexible peptide section Gly
4During Ser, its aminoacid sequence is shown in SEQ ID NO:7.
Linker is flexible peptide section Ala
3Ser
2The time, its aminoacid sequence is shown in SEQ ID NO:8.
Wherein, said fusion rotein is the fusion rotein that 2 Exendin-4-Linker and human IgG1 Fc Fc two mutants reassemble into; Linker is flexible peptide section (Gly
4Ser) n, n=1-5; The amino acid whose sudden change position of IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to the EU numbering system.
Through the flexible polypeptied chain of Linker the Exendin-4 dimer is connected with IgG1 Fc two mutants, has at utmost kept the BA of Exendin-4, the while has at utmost been reduced the untoward reaction that the lytic activity of Fc section causes.
Utilize the clear and definite Exendin-4 dimer of action target spot to be connected with human IgG Fc1Fc two mutants, efficiently express this fusion rotein, be used for I type, type II diabetes treatment through engineered method through flexible peptide Linker.Through the effectively effect of prolong drug transformation period of IgG1 Fc two mutants, the untoward reaction of avoiding unnecessary ADCC effect and CDC effect to bring improves patient's compliance, reduces patient's administration number of times, and result of treatment is good.
Its mechanism of action is: Exendin-4 and IgG1 Fc two mutants merge the molecular weight that can effectively improve polypeptide, thereby reduce the filtration of renal glomerulus; IgG Fc section can be through prolonging its transformation period in vivo with intravital FcRn receptors bind simultaneously.
IgG Fc section is to carry out the important activity zone that prolongs this fusion rotein transformation period; This fragment can be come the transformation period of prolong drug with intravital FcRn receptors bind through the mode of recycling; Natural in theory IgG Fc (four hypotypes) all has this function; Yet because natural IgG Fc fragment also has ADCC effect and CDC effect (this effect is crucial usually) on therapeutic antibodies; So the present invention suddenlys change to natural IgG Fc fragment, to reduce the untoward reaction that its ADCC effect and CDC effect are brought.In addition, IgG1 Fc is that use is maximum at present, and research also is the most thorough, and its content in vivo is the highest, thus also be safest aspect drug use, so the present invention adopts human IgG1 Fc to suddenly change.
Wherein, said fusion rotein is the fusion rotein that 2 Exendin-4-Linker and human IgG1 Fc two mutants reassemble into; Wherein, the Linker that is connected with Exendin-4 respectively of two ends is flexible peptide section (Gly
4Ser) n, n=3; Wherein, the Linker that an end is connected with Exendin-4 and the other end is connected with human IgG1 Fc two mutants is flexible peptide section (Gly
4Ser) n, n=1; Wherein, the amino acid whose sudden change position of IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to the EU numbering system.
The nucleotide sequence of this fusion rotein Exendin-4-Linker-Exendin-4-Linker-IgG1 Fc two mutants is shown in SEQ ID NO:9.
The aminoacid sequence of this fusion rotein is shown in SEQ ID NO:10.
Wherein, said Expression of Fusion Protein carrier is pOptiVEC-TOPO, and said fusion rotein is expressed in mammalian cell, and said cell is selected from Chinese hamster ovary celI, HeLa cell, bhk cell.POptiVEC-TOPO TA cloning Kit (Invitrogen product, catalog no.12744-017).
Preferably, said Chinese hamster ovary celI is CHO DG44 cell (Invitrogen product, catalog no.12609-012).
Wherein, said fusion rotein is in the application of treatment I type, type II diabetes.Utilize engineered method to prepare a kind of have the treatment I type of obvious long-actingization characteristic, the recombinant protein drug of type II diabetes; This medicine can reduce diabetics's blood sugar and glycolated hemoglobin (HbA1c) effectively; Obviously prolong its transformation period simultaneously; Draft all medications once, have better compliance, can reduce patient's psychology and physiological load than the diabetes medicament on the existing market (inject 1-2 every day).This fusion rotein has the Fc fragment of sudden change simultaneously, can at utmost reduce ADCC effect and CDC effect that natural IgG Fc is brought, thereby effectively reduces the untoward reaction of medicine.This invention compared with prior art has higher security and practicality.Compare with other mellitus class medicines and to have a lot of advantages: 1. can significantly reduce HbAlC level and glucose level control; 2. because its adjusting function relies on glucose level, therefore can not cause risk of hypoglycemia; 3. strengthen satietion, reduce excessive absorption of food and fat-reducing; 4. improve the β cell function; 5. control the generation of postprandial hyperglycemia.
For realizing above-mentioned another purpose, the present invention adopts following technical scheme.
A kind of preparation method who is used to treat the fusion rotein of mellitus is provided; This method is included in the host cell of cultivating claim 6 under the condition that is suitable for expressing this fusion rotein; Host cell through cell cultures, centrifugal after, supernatant is used affinity chromatography, ion exchange chromatography and sieve chromatography purifying respectively; What wherein, use in the affinity chromatography is the affine filler of rProtein A Sepharose FF; What wherein, use in the ion exchange chromatography is Q Sepharose FF filler; What wherein, use in the sieve chromatography is Superdex 200 fillers.
Particularly, the preparation method of this fusion rotein may further comprise the steps:
The acquisition of step (1) goal gene: adopt full gene synthetic mode to obtain goal gene DiExendin-4-Ig the dna sequence dna of Exendin-4, the dna sequence dna of Linker and the dna sequence dna of IgG1 Fc two mutants;
Step (2) is cloned on the suitable expression vector: after goal gene DiExendin-4-Ig is synthetic, behind pcr amplification, it is cloned among the expression vector pOptiVEC-TOPO;
Step (3) transfection proper host cell, express: host cell is a CHO DG44 cell, treat PCR, double digestion and order-checking identify correct after, a large amount of amplifications of correct clone are gone forward side by side line linearityization to carry out CHO DG44 cell transfecting;
Step (4) separation and purification target protein: through expressing the purifying process that supernatant pre-treatment, affinity chromatography, ion exchange chromatography and sieve chromatography combine, detect through SDS-PAGE and HPLC successively, its purity is greater than 98%.
All with reference to " operational manual " of " molecular cloning experiment guide " and related prods, worker etc. gives birth to like NEB, invitrogen, Shanghai to the technique means that adopts among the present invention in the biological reagent company that reagent all adopts this field to approve.The dna sequence dna of the dna sequence dna of the dna sequence dna of Exendin-4, Linker and IgG1 Fc two mutants all adopts full gene synthetic mode to obtain among the present invention, and has carried out codon optimized being beneficial to and in host cell CHO DG44 cell, efficiently express.
Beneficial effect of the present invention is:
A kind of fusion rotein that is used to treat mellitus of the present invention, fusion rotein are the fusion rotein that N Exendin-4-Linker and human IgG Fc two mutants reassemble into; Linker is one group of flexible peptide section of being made up of hydrophobic amino acid, and its amino acid whose number is less than 25; Human IgG Fc two mutants is IgG1 Fc, IgG2 Fc or IgG4 Fc two mutants; N is 2-8.Through the flexible polypeptied chain of Linker Exendin-4 is connected with IgG1 Fc two mutants; The BA that has at utmost kept Exendin-4, it not only has Exendin-4 can stimulate secretion of insulin, suppress the biological activity of the release of hyperglycemic-glycogenolytic factor after the meal, has at utmost reduced simultaneously the untoward reaction that the lytic activity of Fc section causes; Have the advantages that to prolong its transformation period in serum; Be used for the treatment of I type and type II diabetes, patient can all medications once, compliance is good; Effectively reduce patient's psychology and physiological load, safe in utilization and practical.
The preparation method who is used to treat the fusion rotein of mellitus of the present invention; This method is included in the host cell of cultivating claim 6 under the condition that is suitable for expressing this fusion rotein; Host cell through cell cultures, centrifugal after, supernatant is used affinity chromatography, ion exchange chromatography and sieve chromatography purifying respectively; What wherein, use in the affinity chromatography is the affine filler of rProtein A Sepharose FF; What wherein, use in the ion exchange chromatography is Q Sepharose FF filler; What wherein, use in the sieve chromatography is Superdex 200 fillers.
Technical maturity of the present invention helps realizing the industrialization production and selling, and the fusion rotein that makes not only has Exendin-4 can be stimulated secretion of insulin, suppress the biological activity of the release of hyperglycemic-glycogenolytic factor after the meal; Also have the characteristics that prolong its transformation period in serum; Be used for the treatment of I type and type II diabetes, patient can all medications once, compliance is good; Effectively reduce patient's psychology and physiological load, safe in utilization and practical.
The invention has the advantages that: (1) thus the present invention suddenlys change to modify to the IgG Fc fragment gene that merges and reduces ADCC effect, CDC effect, reduce its lytic activity, the untoward reaction of effectively avoiding natural IgG Fc to bring.Improve patient's compliance, reduce patient's administration number of times.
(2) the present invention utilize the clear and definite mechanism of action target spot clearly poly Exendin-4 be connected with human IgG Fc two mutants through flexible peptide Linker, efficiently express this fusion rotein through engineered method, be used for the I type, type II diabetes is treated.The BA that has at utmost kept Exendin-4; It not only has Exendin-4 can stimulate secretion of insulin, suppress the biological activity of the release of hyperglycemic-glycogenolytic factor after the meal; Also have the characteristics that prolong its transformation period in serum, be used for the treatment of I type and type II diabetes.
(3) the invention a kind of genetically engineered recombinant protein medicine to treating diabetes with obvious long-actingization characteristic; This medicine can reduce diabetics's blood sugar and glycolated hemoglobin (HbA1c) effectively; The transformation period that has obvious prolongation simultaneously; Draft all medications once, have better compliance, can reduce patient's psychology and physiological load than the diabetes medicament on the existing market (inject 1-2 every day).
(4) preparation method who is used to treat the fusion rotein of mellitus of the present invention, technical maturity, raw material sources are extensive, help accomplishing scale production and selling, and have social value widely, and economic worth is high, production safety and practical.
(5) fusion rotein that the present invention makes is used to treat mellitus has: 1. can significantly reduce HbAlC level and glucose level control; 2. because its adjusting function relies on glucose level, therefore can not cause hypoglycemic danger; 3. strengthen satietion, reduce excessive absorption of food and fat-reducing; 4. improve the β cell function; 5. control the generation of postprandial hyperglycemia.
Description of drawings
Fig. 1 is the structure and the major function district figure of IgG molecule.
Fig. 2 is the structure diagram of four kinds of fusion roteins of a kind of fusion rotein that is used to treat mellitus of the present invention.
Fig. 3 is a kind of pOptiVEC-DiExendin-4-Ig plasmid PCR checking product electrophorogram that is used to treat the fusion rotein of mellitus of the present invention, wherein, and 1:DL2000 Marker; 2: negative control (not adding plasmid); 3-10 is respectively the PCR result of coding 1-8 plasmid.
Fig. 4 is a kind of plasmid pOptiVEC-DiExendin-4-Ig double digestion electrophoresis result figure that is used to treat the fusion rotein of mellitus of the present invention, wherein, and 1:DL2000 Marker; 2:1kbp ladder marker; 3:pOptiVEC-DiExendin-4-Ig is digested plasmid not; 4-11 is numbered 1-8 plasmid double digestion result.
Fig. 5 is a kind of reorganization DiExendin-4-Ig fusion rotein HPLC purity check figure that is used to treat the fusion rotein of mellitus of the present invention.
Fig. 6 is a kind of reorganization DiExendin-4-Ig fusion rotein SDS-PAGE analysis that is used to treat the fusion rotein of mellitus of the present invention, is followed successively by albumen MARKER, reduction electrophoresis and non-reduced electrophoresis from left to right.
Fig. 7 is that a kind of reorganization DiExendin-4-Ig fusion rotein that is used to treat the fusion rotein of mellitus of the present invention stimulates RIN-5F cell cAMP generation figure.
Fig. 8 is 1st day the influence of the fusion rotein DiExendin-4-Ig of a kind of fusion rotein that is used to treat mellitus of the present invention to normal ICR mouse blood sugar TG-AUC.
Fig. 9 is 2nd day the influence of the fusion rotein DiExendin-4-Ig of a kind of fusion rotein that is used to treat mellitus of the present invention to normal ICR mouse blood sugar TG-AUC.
Figure 10 is 3rd day the influence of the fusion rotein DiExendin-4-Ig of a kind of fusion rotein that is used to treat mellitus of the present invention to normal ICR mouse blood sugar TG-AUC.
Figure 11 is 4-6 days the influence of the fusion rotein DiExendin-4-Ig of a kind of fusion rotein that is used to treat mellitus of the present invention to normal ICR mouse blood sugar TG-AUC.
Figure 12 is 7-9 days the influence of the fusion rotein DiExendin-4-Ig of a kind of fusion rotein that is used to treat mellitus of the present invention to normal ICR mouse blood sugar TG-AUC.
Figure 13 is 10-14 days the influence of the fusion rotein DiExendin-4-Ig of a kind of fusion rotein that is used to treat mellitus of the present invention to normal ICR mouse blood sugar TG-AUC.
Figure 14 is 16-20 days the influence of the fusion rotein DiExendin-4-Ig of a kind of fusion rotein that is used to treat mellitus of the present invention to normal ICR mouse blood sugar TG-AUC.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further described.
As shown in Figure 2, in the present embodiment, the fusion rotein that is used to treat mellitus is the fusion rotein that 2 Exendin-4-Linker and human IgG Fc two mutants reassemble into; The flexible peptide section of Linker is (Gly
4Ser) n, n=1-5; Human IgG Fc two mutants is an IgG1 Fc two mutants, and the amino acid whose sudden change position of IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to the EU index
Wherein, the Linker that is connected with Exendin-4 respectively of two ends is flexible peptide section (Gly
4Ser) n, n=3;
Wherein, the Linker that an end is connected with Exendin-4 and the other end is connected with human IgG1 Fc Fc two mutants is flexible peptide section (Gly
4Ser) n, n=1.
As shown in Figure 2, in the present embodiment, the fusion rotein that is used to treat mellitus is the fusion rotein that 2 Exendin-4-Linker and human IgG Fc two mutants reassemble into; The flexible peptide section of Linker is (Gly
4Ser) n, n=1-5; Human IgG Fc two mutants is the IgG1 two mutants, and the amino acid whose sudden change position of IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to the EU index.
Wherein, the Linker that is connected with Exendin-4 respectively of two ends is flexible peptide section (Gly
4Ser) n, n=4;
Wherein, the Linker that an end is connected with Exendin-4 and the other end is connected with human IgG1 Fc two mutants is flexible peptide section (Gly
4Ser) n, n=2.
As shown in Figure 2, in the present embodiment, the fusion rotein that is used to treat mellitus is the fusion rotein that 2 Exendin-4-Linker and human IgG Fc two mutants reassemble into; The flexible peptide section of Linker is (Gly4Ser) n, n=1-5; Human IgG Fc two mutants is the IgG1 two mutants, and the amino acid whose sudden change position of IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to EU index
.
Wherein, the Linker that is connected with Exendin-4 respectively of two ends is flexible peptide section (Gly
4Ser) n, n=3;
Wherein, the Linker that an end is connected with Exendin-4 and the other end is connected with human IgG1 Fc two mutants is flexible peptide section (Gly
4Ser) n, n=3.
As shown in Figure 2, in the present embodiment, the fusion rotein that is used to treat mellitus is the fusion rotein that 2 Exendin-4-Linker and human IgG Fc two mutants reassemble into; The flexible peptide section of Linker is (Gly
4Ser) n, n=1-5; Human IgG Fc two mutants is the IgG1 two mutants, and the amino acid whose sudden change position of IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to the EU index.
Wherein, the Linker that is connected with Exendin-4 respectively of two ends is flexible peptide section (Gly
4Ser) n, n=5;
Wherein, the Linker that an end is connected with Exendin-4 and the other end is connected with human IgG1 Fc two mutants is flexible peptide section (Gly
4Ser) n, n=3.
In the present embodiment, the fusion rotein that is used to treat mellitus is the fusion rotein that 2 Exendin-4-Linker and human IgG Fc two mutants reassemble into; The flexible peptide section of Linker is (Gly
4Ser) n, n=1-5; Human IgG Fc two mutants is the IgG1 two mutants, and the amino acid whose sudden change position of IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to the EU index.
In the present embodiment, the fusion rotein that is used to treat mellitus is the fusion rotein that 2 Exendin-4-Linker and human IgG Fc two mutants reassemble into; The flexible peptide section of Linker is (Ala
3Ser
2) n, n=1-5; Human IgG Fc two mutants is the IgG1 two mutants, and the amino acid whose sudden change position of IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to the EU numbering system.
In the present embodiment, the fusion rotein that is used to treat mellitus is the fusion rotein that 4 Exendin-4-Linker and human IgG Fc two mutants reassemble into; The flexible peptide section of Linker is (Gly
4Ser) n, n=1-5; Human IgG Fc two mutants is the IgG1 two mutants, and the amino acid whose sudden change position of IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to the EU index.
In the present embodiment, the fusion rotein that is used to treat mellitus is the fusion rotein that 8 Exendin-4-Linker and human IgG Fc two mutants reassemble into; The flexible peptide section of Linker is (Gly
4Ser) n, n=1-5; Human IgG Fc two mutants is the IgG1 two mutants, and the amino acid whose sudden change position of IgG1 Fc two mutants is Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236; The combinatorial mutagenesis position of Ala327Gly/Ala330Ser/Pro331Ser, the Position Number that wherein suddenlys change is according to the EU index.
A kind of preparation method who is used to treat the fusion rotein of mellitus; This method is included in the host cell of cultivating claim 6 under the condition that is suitable for expressing this fusion rotein; Host cell through cell cultures, centrifugal after, supernatant is used affinity chromatography, ion exchange chromatography and sieve chromatography purifying respectively; What wherein, use in the sieve chromatography is Superdex 200 fillers.What wherein, use in the affinity chromatography is the affine filler of rProtein A Sepharose FF; What wherein, use in the ion exchange chromatography is Q Sepharose FF filler.
Particularly, the preparation method of this fusion rotein may further comprise the steps:
The acquisition of step (1) goal gene: adopt full gene synthetic mode to obtain goal gene DiExendin-4-Ig the dna sequence dna of Exendin-4, the dna sequence dna of Linker and the dna sequence dna of IgG1 Fc two mutants;
Step (2) is cloned on the suitable expression vector: goal gene DiExendin-4-Ig is synthetic after behind the pcr amplification, it is cloned among the expression vector pOptiVEC-TOPO;
Step (3) is transfected in the proper host cell to be expressed: host cell is a CHO DG44 cell, treat PCR, double digestion and order-checking identify correct after, a large amount of amplifications of correct clone are gone forward side by side line linearityization to carry out the transfection of CHO DG44 cell;
Step (4) separation and purification target protein: through expressing the purifying process that supernatant pre-treatment, affinity chromatography, ion exchange chromatography and sieve chromatography combine, detect through SDS-PAGE and HPLC successively, its purity is greater than 98%.
The design of experimental example 1 reorganization DiExendin-4-Ig fusion rotein and the structure of expression vector pOptiVEC-DiExendin-4-Ig
1. the design of reorganization DiExendin-4-Ig fusion rotein.
The present invention according to mammalian cell access to your password the son preferences; Known Exendin-4 fragment and the segmental aminoacid sequence of sudden change back human IgG1 Fc; Designed one section new reorganization DiExendin-4-Ig fusion rotein, the nucleotide sequence of this fusion rotein is shown in SEQ ID NO:11.
Wherein preceding 10 bit bases are KOZAK sequences; The 11-67 bit base is the signal peptide codon of mouse Ig heavy chain; 68-184,230-346 bit base are the Exendin-4 codon; 185-229,347-361 bit base are the connection peptides codon, and 362-1039 is that base is a human IgG1 Fc two mutants codon, and last three bit bases are terminator codon.
The aminoacid sequence of this fusion rotein is shown in SEQ ID NO:12, and wherein preceding 19 amino acid (line part) are signal peptide sequence.
The above-mentioned sequence that designs is adopted full gene synthetic method preparation.After synthetic the completion, according to following PCR system amplifying target genes.
100 μ l PCR reaction systems:
| Reagent | Content |
| Polynucleotide passage (2 μ g/ μ l) | 1 |
| 10 * PCR reacts |
10 μl |
| 2.5 mM |
8 μl |
| Primer 1 (100 mM, SEQ ID NO:13) | 0.2 μl |
| Primer 2 (100 mM, SEQ ID NO:14) | 0.2 μl |
| Platinum Taq polymerase | 0.4 μl |
| Sterile water | 80.2 μl |
Reaction conditions:
Sex change in advance: 94 ℃, 2 minutes;
Major cycle: 94 ℃, 30 seconds, 55 ℃, 30 seconds, 72 ℃, 1 minute;
Cycle number: 30;
Extend the back: 72 ℃, and 10 minutes.
2. electrophoresis is identified with gel and is reclaimed.
After carrying out PCR according to above-mentioned condition, get 5 μ l products and on 0.8% agarose gel electrophoresis, identify, find the band (as shown in Figure 3) about a unique molecular weight 1000bp in the product.Carry out glue with QIAGEN QIAquick Gel Extraction Kit and reclaim purifying, obtain being about 2.5 μ g target dnas at last.
3. the structure of reorganization DiExendin-4-Ig fusion protein expression vector.
Operate according to pOptiVEC-TOPO vector specification sheets; Above-mentioned dna fragmentation is cloned on this carrier; After transforming One Shot Chemically Competent E. coli; Get 8 single colony inoculations in containing 100 μ g/ml penbritin LB substratum, 37 ℃, the 250rpm amplification cultivation of spending the night is with plasmid extraction purification kit (TIANGEN) extracting DNA and carry out enzyme and cut and select 2 correct clones (as shown in Figure 4) of size after the evaluation and carry out dna sequencing.Sequencing result shows that 2 clones are all correct, i.e. pOptiVEC-DiExendin-4-Ig.
Experimental example 2 reorganization DiExendin-4-Ig Expression of Fusion Protein
1. the extraction of expression vector pOptiVEC-DiExendin-4-Ig.
Get and identify No. 1 correct plasmid bacterial strain, be inoculated in 500ml and contain in the LB substratum of penbritin, 37 ℃, 250rpm were cultivated 18 hours.With plasmid extraction purification kit (TIANGEN) extracting DNA, extractive process is carried out according to the test kit specification sheets that producer provides.Extracting is carried out the linearizing of Pvu I single endonuclease digestion with plasmid after accomplishing, in order to follow-up transfection.
2.CHO the transfection of DG44 cell and expression.
Adopt FreeStyle MAX transfection reagent transfection CHO cell, CHO DG44 cell and transfection reagent are all available from Invitrogen company.The transfection schedule of operation is carried out according to the specification sheets that producer provides.
Cell after the transfection is through the CD of HT (-) OptiCHO
TMPerfect medium cultured continuously one month to be obtaining positive transfection clone, and then with the CD OptiCHO that contains methotrexate (MTX, working concentration are 100-500nM)
TMPerfect medium pressurization screening, this process needs two months.Then stable positive colony is carried out the mono-clonal screening through limiting dilution assay; With semisolid medium CloneMedia-CHO (Genetix Company products; Cat no K8710) cell suspension is adjusted to 1cell/100 μ L/ hole and inoculates in 96 orifice plates, place 37 ℃, 8%CO
2Cultivate in the incubator about 3 weeks, treat the monoclonal cell amplification after, with Expression of Fusion Protein amount in the ELISA method detection supernatant.The guarantor plants after choosing 3 the highest clonal expansions of expression, and getting wherein, F10 number clone carries out amplification culture, expression.
This part operation is fully according to operational manual (the pOptiVEC-TOPO-TA Cloning Kit of invitrogen company; Cat nos. 12744-017; 12745-014,12762-019), wherein used main agents also all is selected from invitrogen company.
The purifying of experimental example 3 reorganization DiExendin-4-Ig fusion roteins
Because this fusion rotein has IgG Fc fragment; Preferably can carry out affinity purification and obtain target protein, in this embodiment, the purifying process that we have adopted affinity chromatography, ion exchange chromatography and sieve chromatography to combine; Detect through SDS-PAGE and HPLC, its purity can 98%.Concrete grammar is following:
1. express the supernatant pre-treatment:
NaOH with 1M slowly regulates pH to 7.3 with supernatant, at 4 ℃ of centrifugal 10min of following 4000rpm, collects supernatant.
2. affinity chromatography:
Behind 10 mM PB (pH7.3) damping fluid balance rProtein A-Sepharose F.F. (GE) affinity columns; With appearance on the above-mentioned centrifugal supernatant; Again with same damping fluid washing affinity column to the OD value of 280nm less than 0.01; Citrate buffer solution (pH 4) with 0.1M washes fusion rotein, collects elution peak.Immediately with elution peak with 200 mM Na
2HPO
4It is for use accurately to regulate pH to 7.5.
3. ion exchange chromatography:
Behind 10 mM PB (pH7.5) damping fluid balance SP Sepharose F.F. (GE) chromatography columns; With appearance on the above-mentioned solution; With same damping fluid washing chromatography base for post line, carry out wash-out again, collect elution peak with the phosphoric acid buffer (pH7.5) that contains 100mM NaCl.
4. sieve chromatography:
With 10 mM PB (pH7.0) damping fluid balance Superdex 200 (GE) molecular sieve chromatographies, last appearance is also collected elution peak.Elution peak is stored in-20 ℃.Sampling detects its purity and molecular weight, and purity is greater than 98% as a result, and apparent molecular weight and theoretical molecular are near (like Fig. 5, shown in 6).
Experimental example 4 reorganization DiExendin-4-Ig fusion rotein external activities detect
This embodiment adopts the cAMP enzyme to join the extracorporeal biology activity that assay method detects reorganization DiExendin-4-Ig.The cAMP detection kit of being selected for use is the R&D Company products, and working method is also carried out according to the test kit specification sheets, and clone is rat Langerhans islet knurl RIN-5F cell, and the cultivation of going down to posterity of cell is carried out according to ordinary method, and positive reference substance is Exendin-4.Concrete operation method is following:
The RIN-5F cell is cultivated under 37 ℃, the condition of 5% carbonic acid gas in the high sugared nutrient solution of the RPMI-1640 that contains 10% calf serum, 1mol/L Sodium.alpha.-ketopropionate, 0.5%HEPES (pH7.2).The cell of taking the logarithm vegetative period, trysinization becomes cell suspension, and the adjustment cell is to finite concentration.Adding the cell suspension cultivated 3 days in CO2gas incubator in 96 well culture plates.Remove cells and supernatant, add serum free medium and continue to cultivate 2h.Get trial target and reference substance and with the cell culture fluid that contains certain density IBMX and bovine serum albumin testing sample is made certain proportion dilution, 4-6 extent of dilution altogether respectively.Remove cell conditioned medium, add the trial target and the reference substance of the good different concns of dilution, each extent of dilution is done 3 multiple holes.In CO2gas incubator, place 10-20min, remove cell conditioned medium, detect with the cAMP test kit, the detection wavelength is 405nm, and reference wavelength is 570nm.The result: under equal volumetric molar concentration, DiExendin-4-Ig has higher BA (as shown in Figure 7) than Exendin-4.
Experimental example 5 is set up the ELISA method and is detected the content of expressing DiExendin-4-Ig fusion rotein in the supernatant
With coating buffer dilution Anti human IgG (γ-chain specific) (1:2000), add in the 96 hole enzyme plates, put 4 ℃ and spend the night with 100 μ l/holes; PBS-T washes plate 3 times; 200ul/ml adds in the enzyme plate with the 0.1%BSA confining liquid, puts 37 ℃, 2 hours; PBS-T washes plate 3 times; Standard substance (10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml, 0.3125ng/ml) that dilution is good and expression supernatant add in the entering plate with 100 μ l/ holes, put 37 ℃, 2 hours.PBS-T washes plate 4 times, presses 1:1000 dilution Anti human IgG (whole molecule)-HRP with 0.1%BSA-PBS-T, adds in the entering plate with 100 μ l/holes, puts 37 ℃, 1 hour.PBS-T washes plate 4 times, adds the interim OPD chromogenic substrate of preparing with 100 μ l/hole, room temperature, and lucifuge colour developing 10min adds 1mol/L sulfuric acid 50 μ l termination reactions, detects the A490 value with ELIASA.Do typical curve with standard substance A value and corresponding standard substance concentration,, calculate the content of DiExendin-4-Ig fusion rotein in the supernatant with sample A value to be checked and extension rate.
The preliminary pharmacodynamic analysis of experimental example 6 reorganization DiExendin-4-Ig fusion roteins
Normal ICR mouse is divided into 4 groups according to body weight at random after SPF level Animal House flexibility is raised 3 days, be respectively normal control group (Nor), 1 mg/kg, 2 mg/kg and 4 mg/kg, 7 every group.Each treated animal overnight fasting (freely drinking water), tail point blood sampling next day, subcutaneous injection administration then (0.1 ml/10 g, the normal control group gives saline water).30 min irritate stomach and give glucose (0.1 ml/10 g) after administration, and respectively at 30,60,120 min tails point blood sampling behind the glucose load, measure blood sugar (glucose oxidase method);
After administration the 2nd day and the 3rd day, each treated animal tail point blood sampling back fasting 4 h (freely drink water) irritated stomach then and gives glucose (the same), and takes a blood sample mensuration blood sugar respectively at 30 and 60 min tails behind the glucose load are sharp;
After administration the 4th day to the 14th day, each treated animal fasting 4 h (freely drinking water) back was irritated stomach and is given glucose (the same), and blood sugar is measured in the blood sampling of 30 min tails point behind glucose load.
2. result:
1) influence that normal ICR mouse blood sugar was changed in the 1st day behind the DiExendin-4-Ig single-dose
Compare with the normal control group, can both significantly reduce area (as shown in Figure 8) under the blood glucose curve after normal ICR mouse oral glucose is loaded behind each dosage single-dose of DiExendin-4-Ig sample.
2) influence that normal ICR mouse blood sugar was changed in the 2nd day behind the DiExendin-4-Ig single-dose
Compare with the normal control group; Can also significantly reduce area (as shown in Figure 9) under the blood glucose curve behind non-fasting serum glucose and the oral glucose load of normal ICR mouse behind each dosage single-dose of DiExendin-4-Ig sample on the 2nd day, explain to be at least its action time 1 day.
3) influence that normal ICR mouse blood sugar was changed in the 3rd day behind the DiExendin-4-Ig single-dose
Compare with the normal control group; Can also significantly reduce area (shown in figure 10) under the blood glucose curve behind non-fasting serum glucose and the oral glucose load of normal ICR mouse behind each dosage single-dose of DiExendin-4-Ig sample on the 3rd day, explain to be at least its action time 2 days.
4) influence that normal ICR mouse blood sugar was changed in 4-6 days behind the DiExendin-4-Ig single-dose
Compare with the normal control group; Can both significantly reduce area (shown in figure 11) under the blood glucose curve of non-fasting serum glucose and oral glucose load back 30 min of normal ICR mouse behind each dosage single-dose of DiExendin-4-Ig sample in 4-6 days, explain to be at least its action time 5 days.
5) influence that normal ICR mouse blood sugar was changed in 7-9 days behind the DiExendin-4-Ig single-dose
Compare with the normal control group; Can both significantly reduce area (shown in figure 12) under the blood glucose curve of normal ICR mouse oral glucose load back 30 min behind each dosage single-dose of DiExendin-4-Ig in 7-9 days; But the reduction effect to the non-fasting serum glucose of ICR mouse weakens gradually, explains to be at least its action time 8 days.
6) influence that normal ICR mouse blood sugar was changed in 10-14 days behind the DiExendin-4-Ig single-dose
Compare with the normal control group; Can both significantly reduce area (shown in figure 13) under the blood glucose curve of normal ICR mouse oral glucose load back 30 min behind each dosage single-dose of DiExendin-4-Ig in 10-14 days; To the non-fasting serum glucose of ICR mouse not obviously influence, but still explain and be at least its action time 13 days.
7) influence that normal ICR mouse blood sugar was changed in 16-20 days behind the DiExendin-4-Ig single-dose
Compare with the normal control group; Can both significantly reduce area (shown in figure 14) under the blood glucose curve of normal ICR mouse oral glucose load back 30 min behind the DiExendin-4-Ig 4 mg/kg single-doses in 16-20 days; Explain to be about 19 days its action time, and all be about 15 days the action time of 2 mg/kg and 1 mg/kg.
3. experiment brief summary:
Can both significantly reduce non-fasting serum glucose of normal ICR mouse and the blood sugar behind the oral glucose load behind DiExendin-4-Ig sample 4 mg/kg, 2 mg/kg and the 1 mg/kg single-dose; Has good dose-effect relationship; And 2mg/kg and 1mg/kg action time are 15 days, and 4mg/kg was at least 19 days action time.
Certainly, the above only is a preferred embodiments of the present invention, so all equivalences of doing according to the described structure of patent claim of the present invention, characteristic and principle change or modify, includes in patent claim of the present invention.
Claims (9)
1. a fusion rotein that is used to treat mellitus is characterized in that, said fusion rotein is the fusion rotein that N Exendin-4-Linker and human IgG Fc two mutants reassemble into;
Wherein, Linker is one group of flexible peptide section of being made up of hydrophobic amino acid, and its amino acid whose number is less than 25;
Wherein, human IgG Fc two mutants is IgG1 Fc, IgG2 Fc or IgG4 Fc two mutants;
Wherein, N is 2-8.
2. a kind of fusion rotein that is used to treat mellitus according to claim 1 is characterized in that: said fusion rotein is the fusion rotein that 2 Exendin-4-Linker and human IgG1 Fc two mutants reassemble into.
3. a kind of fusion rotein that is used to treat mellitus according to claim 1 is characterized in that: said human IgG Fc two mutants is an IgG1 Fc two mutants; The amino acid whose sudden change position of IgG1 Fc two mutants is the combinatorial mutagenesis position of among the Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236/Ala327Gly/Ala330Ser/Pro331Ser one sudden change position or at least two positions, and the Position Number that wherein suddenlys change is according to the EU numbering system.
4. a kind of fusion rotein that is used to treat mellitus according to claim 1 is characterized in that: said Linker is flexible peptide section (Gly
4Ser) n, n=1-5 or flexible peptide section (Ala
3Ser
2) n, n=1-5.
5. a kind of fusion rotein that is used to treat mellitus according to claim 2 is characterized in that: said fusion rotein is the fusion rotein that 2 Exendin-4-Linker and human IgG1 Fc two mutants reassemble into;
Wherein, Linker is flexible peptide section (Gly
4Ser) n, n=1-5;
Wherein, the amino acid whose sudden change position of IgG1 Fc two mutants is the combinatorial mutagenesis position of Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236/Ala327Gly/Ala330Ser/Pro331Ser, and the Position Number that wherein suddenlys change is according to the EU numbering system.
6. a kind of fusion rotein that is used to treat mellitus according to claim 5 is characterized in that: said fusion rotein is the fusion rotein that 2 Exendin-4-Linker and human IgG1 Fc two mutants reassemble into;
Wherein, the Linker that is connected with Exendin-4 respectively of two ends is flexible peptide section (Gly
4Ser) n, n=3;
Wherein, the Linker that an end is connected with Exendin-4 and the other end is connected with human IgG1 Fc two mutants is flexible peptide section (Gly
4Ser) n, n=1;
Wherein, the amino acid whose sudden change position of IgG1 Fc two mutants is the combinatorial mutagenesis position of Glu233Pro/Leu234Val/Leu235Ala/ Δ Gly236/Ala327Gly/Ala330Ser/Pro331Ser, and the Position Number that wherein suddenlys change is according to the EU numbering system.
7. a kind of fusion rotein that is used to treat mellitus according to claim 1; It is characterized in that: said Expression of Fusion Protein carrier is pOptiVEC-TOPO; Said fusion rotein is expressed in mammalian cell, and said cell is selected from Chinese hamster ovary celI, HeLa cell and bhk cell.
8. according to any described a kind of fusion rotein that is used to treat mellitus among the claim 1-7, it is characterized in that: said fusion rotein is in the application of treatment I type, type II diabetes.
9. preparation method who is used to treat the fusion rotein of mellitus; It is characterized in that: this method is included in the host cell of cultivating claim 6 under the condition that is suitable for expressing this fusion rotein; Host cell through cell cultures, centrifugal after, supernatant is used affinity chromatography, ion exchange chromatography and sieve chromatography purifying respectively;
What wherein, use in the affinity chromatography is the affine filler of rProtein A Sepharose FF;
What wherein, use in the ion exchange chromatography is Q Sepharose FF filler;
What wherein, use in the sieve chromatography is Superdex 200 fillers.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100378514A CN102558362A (en) | 2012-02-17 | 2012-02-17 | Fusion protein for treating diabetes and preparation method for fusion protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100378514A CN102558362A (en) | 2012-02-17 | 2012-02-17 | Fusion protein for treating diabetes and preparation method for fusion protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102558362A true CN102558362A (en) | 2012-07-11 |
Family
ID=46405090
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100378514A Pending CN102558362A (en) | 2012-02-17 | 2012-02-17 | Fusion protein for treating diabetes and preparation method for fusion protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102558362A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232666A (en) * | 2014-09-01 | 2014-12-24 | 江苏海王生物制药有限公司 | Gene expressing recombinant exenatide and carrier thereof |
| CN105452481A (en) * | 2013-06-07 | 2016-03-30 | 麻省理工学院 | Affinity-based detection of ligand-encoded synthetic biomarkers |
| CN106046176A (en) * | 2016-08-16 | 2016-10-26 | 中国药科大学 | High-activity long-acting blood-glucose-reducing fusion protein, preparation method and medicinal application thereof |
| US9970941B2 (en) | 2009-03-02 | 2018-05-15 | Massachusetts Institute Of Technology | Methods and products for in vivo enzyme profiling |
| US11054428B2 (en) | 2018-03-05 | 2021-07-06 | Massachusetts Institute Of Technology | Inhalable nanosensors with volatile reporters and uses thereof |
| US11428689B2 (en) | 2016-05-05 | 2022-08-30 | Massachusetts Institute Of Technology | Methods and uses for remotely triggered protease activity measurements |
| US11448643B2 (en) | 2016-04-08 | 2022-09-20 | Massachusetts Institute Of Technology | Methods to specifically profile protease activity at lymph nodes |
| US11519905B2 (en) | 2017-04-07 | 2022-12-06 | Massachusetts Institute Of Technology | Methods to spatially profile protease activity in tissue and sections |
| US11549947B2 (en) | 2011-03-15 | 2023-01-10 | Massachusetts Institute Of Technology | Multiplexed detection with isotope-coded reporters |
| US11835522B2 (en) | 2019-01-17 | 2023-12-05 | Massachusetts Institute Of Technology | Sensors for detecting and imaging of cancer metastasis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525386A (en) * | 2008-03-05 | 2009-09-09 | 浙江华阳药业有限公司 | Fusion protein of Exendin-4 tandem polypeptide and human serum albumin, preparation and application thereof |
| CN101891823A (en) * | 2010-06-11 | 2010-11-24 | 北京精益泰翔技术发展有限公司 | Exendin-4 and analog fusion protein thereof |
-
2012
- 2012-02-17 CN CN2012100378514A patent/CN102558362A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525386A (en) * | 2008-03-05 | 2009-09-09 | 浙江华阳药业有限公司 | Fusion protein of Exendin-4 tandem polypeptide and human serum albumin, preparation and application thereof |
| CN101891823A (en) * | 2010-06-11 | 2010-11-24 | 北京精益泰翔技术发展有限公司 | Exendin-4 and analog fusion protein thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《Eur. J. Immunol.》 19991231 Kathryn L. Armour et al. Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities 第2613-2624页 1-9 第29卷, * |
| KATHRYN L. ARMOUR ET AL.: "Recombinant human IgG molecules lacking Fcγ receptor I binding and monocyte triggering activities", 《EUR. J. IMMUNOL.》, vol. 29, 31 December 1999 (1999-12-31), pages 2613 - 2624 * |
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11703510B2 (en) | 2009-03-02 | 2023-07-18 | Massachusetts Institute Of Technology | Methods and products for in vivo enzyme profiling |
| US9970941B2 (en) | 2009-03-02 | 2018-05-15 | Massachusetts Institute Of Technology | Methods and products for in vivo enzyme profiling |
| US11549951B2 (en) | 2009-03-02 | 2023-01-10 | Massachusetts Institute Of Technology | Methods and products for in vivo enzyme profiling |
| US10883998B2 (en) | 2009-03-02 | 2021-01-05 | Massachusetts Institute Of Technology | Methods and products for in vivo enzyme profiling |
| US11549947B2 (en) | 2011-03-15 | 2023-01-10 | Massachusetts Institute Of Technology | Multiplexed detection with isotope-coded reporters |
| CN105452481A (en) * | 2013-06-07 | 2016-03-30 | 麻省理工学院 | Affinity-based detection of ligand-encoded synthetic biomarkers |
| US10527619B2 (en) | 2013-06-07 | 2020-01-07 | Massachusetts Institute Of Technology | Affinity-based detection of ligand-encoded synthetic biomarkers |
| US11977074B2 (en) | 2013-06-07 | 2024-05-07 | Massachusetts Institute Of Technology | Affinity-based detection of ligand-encoded synthetic biomarkers |
| CN104232666A (en) * | 2014-09-01 | 2014-12-24 | 江苏海王生物制药有限公司 | Gene expressing recombinant exenatide and carrier thereof |
| US11448643B2 (en) | 2016-04-08 | 2022-09-20 | Massachusetts Institute Of Technology | Methods to specifically profile protease activity at lymph nodes |
| US11428689B2 (en) | 2016-05-05 | 2022-08-30 | Massachusetts Institute Of Technology | Methods and uses for remotely triggered protease activity measurements |
| WO2018032929A1 (en) * | 2016-08-16 | 2018-02-22 | 中国药科大学 | Highly active, long-acting anti-diabetic fusion protein, and manufacturing method and pharmaceutical application thereof |
| US11078250B2 (en) | 2016-08-16 | 2021-08-03 | China Pharmaceutical University | High-activity long-acting hypoglycemic fusion protein as well as preparation method and medical application thereof |
| CN106046176B (en) * | 2016-08-16 | 2019-09-10 | 中国药科大学 | A kind of high active long-acting hypoglycemic fusion protein and preparation method thereof and medical usage |
| CN106046176A (en) * | 2016-08-16 | 2016-10-26 | 中国药科大学 | High-activity long-acting blood-glucose-reducing fusion protein, preparation method and medicinal application thereof |
| US11519905B2 (en) | 2017-04-07 | 2022-12-06 | Massachusetts Institute Of Technology | Methods to spatially profile protease activity in tissue and sections |
| US11054428B2 (en) | 2018-03-05 | 2021-07-06 | Massachusetts Institute Of Technology | Inhalable nanosensors with volatile reporters and uses thereof |
| US11835522B2 (en) | 2019-01-17 | 2023-12-05 | Massachusetts Institute Of Technology | Sensors for detecting and imaging of cancer metastasis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102558362A (en) | Fusion protein for treating diabetes and preparation method for fusion protein | |
| AU2020277290A1 (en) | Composition for treating diabetes mellitus comprising insulin and a GLP-1/glucagon dual agonist | |
| CN103957926B (en) | The engineered polypeptide of acting duration and the immunogenicity reduced with enhancing | |
| CN106220724B (en) | 21 recombinant protein of human fibroblastic growth factor and its preparation method and application | |
| EP2502939B1 (en) | Nonnatural collagen-like protein and use thereof | |
| CN106986940A (en) | Engineered polypeptide with enhanced acting duration | |
| TW201420606A (en) | Homodimeric proteins | |
| CN106432509B (en) | A kind of 21 fusion protein of recombinant human fibroblast growth factor and its preparation method and application for treating metabolic disease | |
| CN102153653B (en) | Fusion protein of tumor blood vessel targeted polypeptide and tissue factor and preparation method thereof | |
| CN1935846A (en) | Fusion protein for treating diabetes, and its preparing method and use | |
| CN102875683B (en) | Fc fusion protein of long-acting recombinant human growth hormone | |
| CN107033234B (en) | Acylated glp-1 derivatives | |
| US20230076768A1 (en) | IL2 Orthologs and Methods of Use | |
| WO2012063984A1 (en) | Improved iduronate-2-sulfatase and use thereof | |
| CN105367664B (en) | Activate GLP-1 receptor and the preparation of the fusion protein of the difunctional effect of Amylin receptor and application thereof | |
| US20200254108A1 (en) | Long-acting single-chain insulin analog and conjugate thereof | |
| CN102516393A (en) | Insulin mimetic peptide fusion protein, mutant and applications thereof | |
| CN106279430B (en) | Analog fusions of Exendin 4 and its production and use | |
| CN113105561B (en) | Preparation method and application of double-target fusion protein | |
| WO2021146485A2 (en) | Cd122 with altered icd stat signaling | |
| CN104558198A (en) | Preparation method and application of fusion protein of GLP-1 analogue and amylin analogue | |
| CN105884901B (en) | Tool persistently controls recombination human serum albumin/glicentin class peptide fusion protein of blood-sugar content function | |
| US20220298215A1 (en) | Multi-receptor (GLP-1 receptor, GIP receptor, and Gcg receptor) agonist protein | |
| WO2022117044A1 (en) | Glp-1/gcg dual receptor agonist polypeptide and fusion protein thereof | |
| CN109776653B (en) | Human serum albumin adhesion peptide and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120711 |